Disease diagnosis and therefore treatment rely upon observations of tissue and cellular characteristics, such as proliferation, cellular and nuclear morphology, vascularization, and specific biomarkers. For example, both the choice of treatment and the risks taken during surgical resection of a tumor mass in the brain are dictated by tumor type and malignancy grade. Tangentially, detailed pathology characterization is revealing considerable overlap in the molecules involved in one disease and the next, consistent with disease continuum rather than discrete disease states. For instance, the lines between Alzheimer's, Pick's, and Parkinson's diseases are often blurred (Galpern and Lang (2006) Ann Neurol 59:449), as are the lines between gradations of a given tumor type (see Caprioli (2005) Cancer Res 65:10642; Schwartz et al. (2005) Cancer Res 65:7674; Iwadate et al. (2004) Cancer Res 64:2496). As the list of overlapping molecules involved in cancers and other diseases grows, so does the need for comprehensive molecular profiling as part of diagnosis (Schwartz, ibid; Schwartz et al. (2004) Clin Cancer Res 10:981). The recent adaptation of mass spectrometers and their respective computer applications to accommodate tissue analysis (Stoeckli et al. (2001) Nat Med 7:493) provides such a comprehensive molecular profile (Schwartz, ibid; Iwadate, ibid; Rahman et al. (2005) Am J Respir Crit Care Med 172:1556; Yanagisawa et al. (2003) Lancet 362:433; Chaurand et al. (2006) Curr Opin Biotechnol 17:431) and saves time compared to histology.
Mass spectrometry (MS) is a well-established technique used to characterize analytes by determining their molecular weight. Ordinarily, mass spectrometry involves the steps of: coating a sample presentation apparatus with an analyte, introducing the sample presentation apparatus into the mass spectrometer, volatilizing and ionizing the molecules of the analyte, accelerating the ionized analyte toward a detector by exposing the ions to an electric and/or a magnetic field, and analyzing the data to determine the mass-to-charge ratio of specific analyte ions. If an analyte remains intact throughout this process, data will be obtained that correspond to a molecular weight for the entire intact analyte ion. Typically however, it is also desirable to obtain data corresponding to the molecular weight of various fragments of the analyte.
Matrix-Assisted Laser Desorption Ionization (MALDI) is an ionization technique often used for mass spectrometric analysis of large and/or labile biomolecules, such as nucleotidic and peptidic oligomers, polymers, and dendrimers, as well as for analysis of nonbiomolecular compounds, such as fullerenes (Karas et al. (1987) Int. J. Mass. Spectrom. Ion Processes 78:53; Spengler and Kaufmann, (1992) Analusis 20:91). MALDI is considered a “soft” ionizing technique, in which both positive and negative ions are produced. The technique usually involves depositing a small volume of sample fluid containing an analyte on a substrate comprised of a photon-absorbing “matrix” material selected to enhance desorption performance. See Karas et al. (1988), “Laser Desorption Ionization of Proteins with Molecular Masses Exceeding 10,000 Daltons,” Anal. Chem., 60:2299-2301. Said matrix material is usually a crystalline organic acid that absorbs electromagnetic radiation near the wavelength of the laser. When co-crystallized with analyte, the matrix material assists in the ionization and desorption of analyte moieties. The sample fluid typically contains a solvent and the analyte. Once the solvent has been evaporated from the substrate, the analyte remains on the substrate at the location where the sample fluid has been deposited. Photons from a laser strike the substrate at the location of the analyte and, as a result, ions and neutral molecules are desorbed from the substrate. Prior to the development of MALDI, analysis of biomolecules by mass spectrometry was quite difficult, if not impossible, since no techniques were available that were gentle enough to volatize intact biomolecules without any degradation or fragmentation. MALDI techniques are particularly useful in providing a means for efficiently analyzing a large number of samples. In addition, MALDI is especially useful in the analysis of minute amounts of sample that are provided over a small area of a substrate surface.
Direct mass spectrometry analysis of a tissue sample affords a wealth of chemical information, providing a molecular landscape of a tissue. Unlike current immunohistochemistry methods, which analyze the concentration and distribution of only a single molecule per experiment, MS imaging provides information on hundred of molecules, affording a better correlation between molecular composition and disease pathology, and therefore a more accurate diagnosis. Indeed, the ability to image a sample with the objective to obtain the detailed spatial arrangement of compounds in an ordered target sample such as a slice of tissue using MALDI MS would be of enormous value in biological research. For example, selected ion surface maps of such samples could provide details of compound compartmentalization, site-specific metabolic processing, and selective binding domains for a very wide variety of natural and synthetic compounds.
Recently, mass spectrometry techniques involving laser desorption have been adapted for cellular analysis. For example, U.S. Pat. No. 5,808,300 to Caprioli describes a method for imaging biological samples with MALDI mass spectrometry. This method allows users to measure the distribution of a specific element or small molecule within biological specimens such as tissue slices or individual cells. In particular, the method can be used for the specific analysis of peptides in whole cells, e.g., by obtaining signals for peptides and proteins directly from tissues and blots of tissues. In addition, the method has been used to desorb relatively large proteins from tissues and blots of tissues in the molecular weight range beyond about 80 kiloDaltons. From such samples, hundreds of peptide and protein peaks can be recorded in the mass spectrum produced from a single laser-ablated site on the sample. When a laser ablates the surface of the sample at multiple sites and the mass spectrum from each site is saved separately, a data array is produced, which contains the relative intensity of any given mass at each site. In the MALDI MS imaging experiment (MSI), hundreds of closely spaced MALDI MS spectra are taken in a grid pattern where each spectrum is analogous to a pixel (Gusev et al. (1995) Anal. Chem. 67:4565; Stoeckli et al. (1999) J Am Soc Mass Spectrom 10:67; Caprioli et al. Anal. Chem. (1997) 69:4751). Each pixel contains information on the mass and intensity of hundreds of biomolecules, which can be translated into a spatial map of molecular distribution and abundance. An image of the sample surface can then be constructed for any given molecular weight, effectively representing a compositional map of the sample surface.
Accordingly, in order to perform mass spectrometry imaging, molecules must be transferred from the tissue and into the gas phase. As described above, this transfer requires a laser within the mass spectrometer, and a matrix which is applied to the tissue section. The matrix is a small acid that crystallizes on the sample, and upon absorbing laser energy is vaporized along with molecules from the tissue. For efficient desorption of large molecules (10 kDa and greater) by MALDI, sinapinic acid is a commonly used matrix. The matrix solution most often applied to tissue samples is 20-30 mg sinapinic acid/ml and about 0.1% trifluoroacetic acid (TFA), in a solvent mixture of 50:50 volume ratio of acetonitrile to water. Hence, all current methods of matrix deposition on tissue slices, such as the solution base described, considerably perturb tissue integrity, resulting in the solubilization and extraction of proteins and peptides from the tissue and their subsequent diffusion from biological location (Gusev, ibid.). Delocalization of proteins has a deleterious effect on image quality. Spatial resolution is lost, and the MS image becomes “blurred.” To minimize protein diffusion in the tissue section, practitioners commonly use a method referred to as “spotting.” The goal of spotting is to apply very small droplets of solution, and since proteins can only diffuse within the drop, the “blurring” is limited to smaller area. Spotting involves either manual deposition using a pipette, or using a recently patented acoustic reagent multispotter device (Aerni et al. (2006) Anal. Chem. 78(3):827-34; U.S. Pat. Nos. 6,707,038, 6,809,319, and 6,855,925 all to Ellson et al.). The diameter of the spots is on average 1 mm, at least one thousand times too large for many histology-based diagnoses. Indeed, there are considerable drawbacks to this practice. Current methods of sample preparation associated with this approach: (1) are expensive (the device of Caprioli, Ellson, et al. costs approximately $300,000, requiring significant capital investment); (2) are time consuming (hours vs. minutes to prepare and deposit sample); (3) do not provide images of sufficient resolution (from 0.2 mm for the acoustic reagent multispotter to 1 millimeter resolution for manual deposition currently, whereas 1 micrometer is needed); (4) do not provide a contiguous image or profile of the tissue; and (5) manual spotting is not reproducible.
Another approach used to cover tissue samples with matrix solution without causing considerable protein displacement is referred to as “spraying.” This method consists of applying the matrix solution uniformly on the tissue section using consecutively minimal volumes of solution to minimize the time that the tissue is in the presence of the solvent. The spraying of matrix solution is typically achieved using a nebulizer, paintbrush or modified electrospray source. Although some practitioners precede spraying or spotting by ethanol fixation, the acetonitrile/water based solution of matrix can still dissolve and displace fixed proteins. Following our own attempts to improve spraying using a modified electrospray source (Schwartz et al. (2003) J Mass Spectrom 38:699), we concluded that this method is still not suited for use in a clinical setting. Although good images can be produced, image quality was inconsistent and spatial resolution was operator-dependent.
Notwithstanding its promise, MS imaging has not yet been incorporated as a medical diagnostic, in part due to a lack of facile, reproducible means of tissue preparation. A sample preparation method of matrix deposition for tissue sections is needed for MALDI MS analysis, which preserves the spatial location of proteins during processing. An objective of the present invention and all attendant embodiments for MALDI MS imaging and sample preparation related thereto is to provide medicine with safe diagnostic strategies that are readily accessible to any established pathology or research laboratory.